P753: TARGETING IL-2-INDUCIBLE T CELL KINASE (ITK) TO BLOCK T LYMPHOCYTE ACTIVATION FOR THE TREATMENT OF APLASTIC ANEMIA MURINE MODEL
Authors: Weiwang LI, Ruonan LI, Jin Mao, Lele Zhang, Liwei Fang, Zhen Gao, Hong Pan, Yajing Chu, Weiping Yuan, Jun Shi
Journal: HemaSphere (2023)

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Autoreactive activated T cell-mediated hematopoietic stem/ progenitor cells (HSPCs) destruction is the core point of pathophysiology in aplastic anemia (AA). The clinically available immunosuppressive agents have obvious side effects and poor specificity targeting on T cells. Therefore, novel targeted therapy is a scientific issue with potential implications of AA treatment. IL-2 inducible T-cell kinase (ITK) is essential for downstream response to TCR signal and plays as the rheostat of T-cell function. Aims: We aimed to clarify the effect of ITK targeted inhibition therapy on bone marrow （BM） hematopoietic injury and donor T cell function in AA mice. The expression level of p-ITK in peripheral blood（PB） T cells of AA patients were analyzed to explore the possibility of ITK inhibitor (ITKi) being used in clinical AA treatment. Methods: First, the expression level of p-ITK in AA model donor T cells were analyzed. Next, ITKi were used to analyze their inhibitory effect on T cell activation and proliferation in vitro. Then we evaluated the effects of ITKi on AA mice disease by detecting blood routine, BM nucleated cell count, sternum pathology and survival of AA mice. The function of the remaining hematopoietic cells was evaluated. At the same time, the inhibitory effect of ITKi on the function of donor T cells was clarified by analyzing the ratio of donor T cells in the spleen and BM, and expression of its effector molecules. Finally, we compared the p-ITK expression in PB T cells of AA patients and healthy donors T cells. Results: The p-ITK expression level of donor T cells in the spleen of AA model recipient mice after transplantation on d5 was significantly higher than that before transplantation, and its expression level in d10 BM donor T cells was lower than that in d5, but still higher than before transplantation. Using two ITKis, PCI-32765 and PRN694, to inhibit the ITK kinase activity of mouse T cells in vitro can significantly inhibit the activation and proliferation of T cells. Using PCI-32765 as a treatment to AA mice, the 25mg/kg group can also significantly improve the hemoglobin and platelet level and bone marrow nucleated cell counts in AA mice. Both 25mg/kg and 50mg/kg treatment dose groups could improve the state of hematopoietic vacancy in the BM and prolong the survival of AA mice. Although the proportion and number of HSCs did not increase significantly after the treatment of AA mice with PCI-32765 25mg/kg, the number of clone units generated by BM cells in vitro increased. The results also showed that the 25 mg/kg group could reduce the proportion of donor T cells, but both the two treatment groups failed to reduce the proportion of functional subsets and the expression of IFN-γ in T cells. Further analysis of the inhibitory effect of PCI-32765 50mg/kg on donor T cells in the spleen on d5 after transplantation found that it significantly reduced the expression of granzyme B and perforin in CD8 T cells. Finally, the analysis of the p-ITK levels showed that the expression of p-ITK in both AA patients and healthy donors ‘ T cells increased after activation in vitro, and the level in AA patients before activation was significantly higher than that in healthy donors. Summary/Conclusion: ITK may be involved in T cell activation both in AA mouse model and AA patients. ITKi can alleviate the disease state of AA mice and improve the colony forming ability of residual hematopoietic cells in vitro by inhibiting part of the functions of donor T cells in AA mice. It is suggested that ITK may be a potential target for immunosuppressive therapy for AA patients. Keywords: Aplastic anemia, Targeted therapy, Human lymphocytes